Our program is a follow-up of the 4.5-year work supported by the R01 CA83017 grant from NIH. As a result, a significant level of progress has been achieved, and multiple advanced molecular and biochemical tools, which are required for achieving a detailed understanding of matrix metalloproteinase (MMP) biology, are now designed and tested. We are now ready to reveal in-depth the mechanisms of the pro-tumorigenic, pro- invasive membrane type-1 MMP (MT1-MMP) functionality and its control in cancer. Our data show that MT1- MMP, a key pro-invasive MMP in cancer, performs multiple and sophisticated functions in malignant cells. Because of the cleavage of cell-surface receptors the short-lived MT1-MMP molecules have a long-lasting effect on cell behavior. The several individual structural domains which are distinct from and additional to the catalytic domain play a well-controlled role in the interactions of MT1-MMP with its targets and also in the multiple signaling and regulatory cellular pathways that result from these interactions. Our main hypothesis which is soundly based on our preliminary observations is that the proteolytic and non-proteolytic parameters regulate the resulting functional activity of MT1-MMP in cancer. Our proposed follow-up wide-ranging and comprehensive research program will be focused on the underlying biochemical, genetic, epigenetic and cellular mechanisms which jointly and reciprocally control the activity, trafficking and overall functionality of cellular MT1-MMP. The proposed research combines basic and clinical aspects of MT1-MMP enzymatic and non-enzymatic functions, emphasizing, among other aspects, the contribution of MT1-MMP expressed by malignant cells to cancer progression. Our Specific Aims are: (1) Determine the functional significance of MT1-MMP proteolysis of protein tyrosine kinase 7 (PTK7)/colon carcinoma kinase-4 (CCK-4) in malignancy, (2) Determine the role the individual structural domains play in MT1-MMP's functions, (3) Determine the critical elements of the epigenetic control of the MMP and TIMP family members and selected invasion-promoting and angiogenesis genes in cancer, and (4) Optimize the structure, selectivity and efficiency of small-molecule MT1- MMP inhibitors. Collectively, the expected results should generate a more coherent and unified view on the functioning of MT1-MMP in malignancy and identify new upstream and downstream target proteins and genes involved in the regulation of this MMP functionality. As a result of our proposed studies, a better understanding of the MMP biology will be accomplished and this knowledge will benefit research not only in cancer but also in other fields such as inflammation, fibrosis, and cardiovascular and neurological disease in which MMPs play a significant pathogenic role. We expect that our findings will give us the tools to identify the crucial contribution of MMPs to tumorigenesis leading to improved diagnosis/prognosis methods, and novel and more effective therapies of malignancies. Precise analysis of the MT1-MMP functionality will pave the way for rational design of inhibitory molecules directed against its specific enzymatic and non-enzymatic functions.